Role of Endothelin Receptor B signaling in the genesis of aganglionic megacolon

描述(申请人提供)：肠神经系统(ENS)主要由一群神经脊源性细胞(ENCC)发育而来，这些细胞进入咽部，然后向尾部穿过发育中的肠道。ENCCs在发育中的肠道需要协调其增殖、分化和迁移。这些细胞过程中的任何一种缺陷单独或合并都足以阻止ENCC的进展。当ENCC没有完成穿过结肠的前进时，肠道的一个区域就没有固有的神经，这是先天性巨结肠(HD)的代表。在编码配体(EDN3)或其受体内皮素-B(EDNRB)的基因中发现了一组与HD常见的突变。该受体在ENCC和肠间充质细胞中均有表达。到目前为止，EDNRB介导的HD的确切细胞缺陷和分子机制仍不清楚。为了解决这些问题，我们利用了一种新的条件等位基因，它允许我们仅在神经脊细胞中敲除EDNRB的表达。通过将我们的EDNRB小鼠与WNT1-Cre转基因小鼠配对，我们在神经脊来源的ENCC中特异性地删除了EDNRB。此外，ENCC还表达黄色荧光蛋白，从而可以记录它们的运动。我们的研究揭示了两个以前未被发现的与EDNRB信号缺陷有关的细胞缺陷。首先，空EDNRB胚胎的ENCC进展在神经脊细胞进入肠道后不久被推迟。其次，当空的EDNRB ENCC到达结肠时，它们表现出异常的轨迹和缺陷的迁移。我们的数据表明这两个缺陷是相关的；最初的延迟导致ENCC比正常情况下晚一天进入结肠。我们假设最初的延迟是由肠前神经脊缺陷引起的，随后的后肠推进失败是由于后肠环境的变化造成的。第一个特定的目的是确定在零突变体中导致ENCC进展最初延迟的细胞缺陷。第二个目的是确定ENCC在结肠中有缺陷的迁移机制。第三个具体目的是确定ENCCs移植到无神经节的出生后后肠是否能恢复后肠的协调运动功能。总之，这些研究将确定由于缺乏EDNRB信号而导致HD的细胞缺陷，并确定建立素协调运动功能与移植的空EDNRB ENCC的可行性。公共卫生相关性：我们的目标是阐明先天性巨结肠症的发病机制，并设计一种为无神经节结肠区域提供功能性神经支配的方法。这些研究将使患有先天性巨结肠和其他胃肠动力障碍的患者受益，因为提供功能性神经是比手术切除更好的选择，因为手术切除会给患者留下终身的胃肠道问题。这里获得的信息也将与包括神经元在内的其他组织的移植有关。

DESCRIPTION (provided by applicant): The enteric nervous system (ENS) develops primarily from a cohort of neural crest- derived cells (ENCCs) that enter at the pharynx before advancing caudally through the developing gut. The advance of ENCCs through the developing gut requires their coordination of proliferation, differentiation, and migration. Defects in any of these cellular processes alone or combined are sufficient to stop ENCC advance. When ENCCs do not complete their advance through the colon, a region of the gut is left without intrinsic innervation and is representative of Hirschprung's disease (HD). One set of mutations commonly associated with HD is found in genes encoding the ligand (EDN3), or its receptor endothelin-B (EDNRB). The receptor is expressed in both ENCCs and gut mesenchyme. To date, the precise cellular defect and molecular mechanism responsible for EDNRB-mediated HD remain unknown. To address these issues we have utilized a novel conditional floxed allele that allows us to knockout ednrb expression in neural crest cells only. By mating our floxed ednrb mice with Wnt1-Cre transgenics we delete ednrb specifically in neural crest-derived ENCC. In addition, the ENCCs also express the yellow fluorescent protein, allowing their movement to be recorded. Our studies have revealed two previously unrecognized cellular defects related to defective EDNRB signaling. First, the ENCC advance in null ednrb embryos is delayed shortly after neural crest cells enter the gut. Second, as null ednrb ENCCs reach the colon, they display aberrant trajectories and defective migration. Our data indicate that these two defects are related; the initial delay results in ENCCs entering the colon a day later than normal. We hypothesize that the initial delay is caused by pre- enteric neural crest defect, and the subsequent failure to advance in the hindgut results from changes in the hindgut environment. The first specific aim is to determine the cellular defect responsible for the initial delay of ENCC advance in the null mutant. The second aim is to determine the mechanism for defective ENCC migration in the colon. The third specific aim is to determine whether ENCCs transplanted into the aganglionic postnatal hindgut can restore coordinated motor function to the hindgut. Together, these studies will identify cellular defects responsible for HD resulting from the absence of EDNRB signaling and determine the feasibility of establishin coordinated motor function with transplanted null ednrb ENCCs. PUBLIC HEALTH RELEVANCE: Our goals are to elucidate the mechanisms underlying Hirschsprung's disease and to devise a method to provide functional innervation to the region of aganglionic colon. These studies will benefit patients with Hirchsprung's disease and other gastrointestinal motility disorders because providing functional innervation is a superior alternative to surgical excision, which leaves patient with lifelonggastrointestinal problems. The information gained here will also be relevant to transplantation of other tissues including neurons.